FR2684381A1 - COMPOUNDS HAVING A PHOSPHORIC AMIDE LINK OR AN ENOL PHOSPHATE LINK FOR THEIR APPLICATION AS A THERAPEUTICALLY ACTIVE SUBSTANCE. - Google Patents

Abstract

A method for treatment of human tumors which are sensitive to the compounds recited below, particularly those which have become resistant to chemotherapy. The method comprises administering an effective amount of creatine phosphate or phosphoenolpyruvic acid.

Description

i Compounds having a phosphoric amide bond or a bond

  Enol phosphate for their application as a therapeutically active substance The present invention relates, for their first application, as a therapeutically active substance, the compounds having an amide bond

  phosphoric or an enol phosphate bond.

  In the context of the present description, the term “compound” means

  having a phosphoric amide bond, any compound whose chemical structure comprises a group: OH

-NH-P = O

  OH A preferred example of such compounds is creatine phosphate, also known as phosphocreatine, which is a substance widely distributed in vertebrates notably present in the striated muscle, but absent from blood and extracellular fluids Another example is arginine phosphate, found in

intervertebrate muscles.

  Likewise, the term “compound having an enol phosphate bond” is understood to mean any compound having in its chemical structure the group: OH

-C-O-P = O

He I

CH 2 OH

  A preferred example of such compounds is phospho-enol-pyruvic acid which is an intermediate endogenous metabolite involved in glycolysis, and which is

  therefore under physiological conditions, localized in cells and absent from the blood.

  The phosphoric amide or enol phosphate bonds of the compounds

  mentioned above are energy-rich bonds.

  It has been discovered, and this constitutes the basis of the present invention, that the compounds exhibiting a phosphoric amide bond or an enol phosphate bond exhibit particularly advantageous pharmacological properties, and

  in particular, a particularly remarkable anti-tumor activity.

  These compounds will therefore find application in particular in the treatment of human tumors, and more particularly tumors from the start or which have become resistant to chemotherapy, such as for example adenocarcinomas (colon, pancreas, stomach, bronchi, kidneys, breasts, uterus, ovaries ); epidermal carcinomas (upper aerodigestive tract, bronchi, urinary tract, anus, skin); melanoma

  malignant, soft tissue sarcomas; leukemia, lymphoma, multiple myeloma.

  Thus, the present invention extends to pharmaceutical compositions, in particular with anti-tumor activity, containing as active ingredient, at least one compound having a phosphoric amide bond, or an enol phosphate bond,

  in combination with a pharmaceutically acceptable excipient.

  These pharmaceutical compositions will generally be prepared according to conventional methods and administered by the venous route, in the form of a bolus or of an intermittent or continuous infusion, or by the intraarterial route, or by the intraperitoneal route, or by the intramuscular route, in doses which can vary from 50 mg to 5 g

  per kilogram of body weight and per 24 hours.

  The invention also aims to cover a process for the preparation of a pharmaceutical composition, in particular with anti-tumor activity, characterized in that it consists in incorporating a pharmaceutically effective amount of at least one compound having a phosphoric amide bond, or a bond enol phosphate, in a

  pharmaceutically acceptable excipient.

  According to a particular characteristic of this process, the composition

  pharmaceutical is formulated as an injectable preparation dosed from 0 1 to 50 g.

  Finally, the present invention also aims to cover a method of treating human tumors, characterized in that it comprises the administration of a therapeutically effective amount of at least one compound having an amide bond

  phosphoric or an enol phosphate bond.

  The pharmacological properties of the compounds having a phosphoric amide bond or an enol phosphate bond have been demonstrated by evaluating the effects of creatine phosphate on cells by various studies.

tumor.

  The observed effects are indisputably due to the presence in this

  molecule of a phosphoric amide bond.

  Further tests on phospho-enol-pyruvic acid suggest that similar pharmacological properties can be obtained from

  of compounds having an enol phosphate bond.

  1 * Method 1.1 Principle The development of an adenocarcinoma of human origin in nude mice is carried out by inoculation of cancer cells Ca CO 2 The effects of creatine phosphate on tumor growth and cell differentiation are studied after administration of the produced intravenously or intraperitoneally The evaluation of these effects is carried out by measuring the size and weight of the tumor and then by histological examination by comparing the data from the control group with those from the treated group. 1.2 Procedure 1.2 1 Preparation of the animal The nude mice, Swiss strain, aged 4 weeks are kept in the isolator for one week before the implantation of the tumor cells in

sterile conditions.

  1.2 2 Cell culture The Ca CO 2 cells are cultured in DMEM medium containing 20% fetal calf serum, 2 mM glutamine and 1% non-essential amino acids, in an incubator with 10% C 02 The culture medium is renewed 3 times a week The

  subculture is carried out using trypsin and EDTA.

  1.2 3 Development of adenocarcinoma of human origin in nude mice After trypsinization, an amount of approximately 10 × 10 6 Ca CO 2 cells (either in 0 2 ml of PBS buffer or in 0 2 ml of culture medium ) are inoculated through

  subcutaneously at the level of the right flank of each host mouse, under sterile conditions.

  The animals are then kept in the isolator.

  The animals are weighed and the tumor is measured with a vernier caliper each day in order to establish a tumor growth curve. The volume of the tumor thus established (hemi-ellipso Ydale) is calculated according to the following formula: volume = 0 5236 x width x length x height (Dethlefsen et ai, 1968) When the animal is sacrificed, the tumor is removed in its entirety and weighed,

  then fixed in 10% formalin or Bouin for anatomopathological examination.

  1.2 4 Study concerning the effect of creatine phosphate on tumor growth and differentiation

  Two series of studies have been carried out with creatine phosphate.

  Each series includes 7 to 8 nude mice divided between control and treated groups. At the start of the study, all the mice had a subcutaneous implantation, on the right flank, of Ca CO 2 tumor cells; Treatment was started 7 days after implantation for experiment 1 and continued for 6 weeks The group of control animals received the vehicle

under the same conditions.

  1 i O For experiment 2, the animals were treated during the exponential phase of tumor growth, namely 6 weeks after the injection of the cells

  tumor and for 3 and a half weeks.

  The dosage was as follows: 0 5 M creatine phosphate dissolved in 0 9% Na CI injected in a volume of 1 ml, at the rate of 6 intraperitoneal injections per week (treated group), 1 ml of physiological serum (Na CI at 0 9%) per injection, at the rate of 6 injections per

week (group of witnesses).

  For the first series, the animals were sacrificed one week after stopping the treatment, and the removed tumors were fixed in 10% formalin. For the second series, the animals were sacrificed when the treatment was stopped, and tumors

samples fixed in the Bouin.

  2 * Results The results are presented in the table below and indicate the weight

  of the tumor in mg, animal by animal.

  WITNESS ANIMALS ANIMALS TREATED BY UP 999-247

  Body weight Tumor weight Body weight Tumor weight (g) (mg) (g) (mg) Start of trial End of trial Start of trial End of trial

17 22 577 2 18 5 23 5 88 9

16.5 23 812 6 20 24 334

) 16 5 21 5 20 5 18 21 84 2

WL 16 22 47 2

27.5 33 2274 24 5 26 287

NOT

8 D 25 31 5 2 240 24 5 29 818

vs

_ 23 27 103 26 28 5 184

23 28 97

READ

21.5 24 10

  ('the average weight gain during treatment is 3 2 g for those treated,

5.0 g for the witnesses.

  The results obtained show that there is no difference in terms of behavior and general condition between the control group and the treated group. A significant difference is observed with regard to the weight of the tumor removed.

  between the two groups, in the two series tested.

  From the histological examination point of view, the samples are identified as poorly differentiated adenocarcinomas. Compared to the neoplasms collected from the control animals, the tumors observed in the treated animals have a very markedly smaller size. No noticeable difference in architecture and

  cytology of the neoplasms observed is not visible.

  3 * Conclusion The results show, depending on the series considered, an inhibition of tumor growth or a tumor regression after administration of creatine

  phosphate in nude mice with established tumors.

TOLERANCE

  Creatine phosphate was administered intravenously once a day for 5 days, at doses 600, 1200, 1800 and 2400 mg / kg, in rats The weight development and feeding behavior of the animals were monitored during the experimental period A biochemical and hematological assessment was carried out on plasma and blood

  collected after animal sacrifice.

  Under the experimental conditions described, creatine phosphate did not induce mortality. The variations observed concern the groups of animals treated with the highest doses studied, which cause a modest increase in transaminases as well as sodium, and a decrease in alkaline phosphatases and of the elements.

figured in blood.

  The other modifications are part of the fluctuations usually

encountered in rats.

  Creatine phosphate has shown excellent biological tolerance for

  doses up to 2400 mg by intravenous administration.

  Similarly, phospho-enol-pyruvic acid showed good biological tolerance and was administered to rats up to a dose of 500 mg / kg without inducing mortality. The following are non-limiting examples of pharmaceutical compositions based on a compound having a phosphoric amine bond or a

enol phosphate bond.

  Creatine phosphate can be presented either in the form of a sterile ready-to-use solution, or in the form of a sterile powder or lyophilisate. It may then be advantageous to use a ballast without pharmacological activity, among

  which we will retain lactose, mannitol.

  The reconstitution solvent may be either chosen from the injection solvents conventionally used (Na CI 9 c, 5% or 15% glucose serum, sorbitol), or

  any other generally available infusion solvent.

  Preferably, the pharmaceutical preparation will be in the form of a sterile lyophilisate containing 25 g of creatine phosphate to be reconstituted

  with 100 ml of a 9 O / o solution of sodium chloride.

Claims (8)

  1 Compounds having an energy-rich bond chosen from a phosphoric amide bond or an enol phosphate bond for their application as   as a therapeutically active substance.   2 Creatine phosphate for its application as a substance therapeutically active.   3 Phospho-enol-pyruvic acid for its application as a substance therapeutically active.   4 Pharmaceutical compositions, characterized in that they contain, as active ingredient, at least one compound having an energy-rich bond chosen from a phosphoric amide bond or a phosphate bond   enol and a pharmaceutically acceptable excipient.   Pharmaceutical compositions with anti-tumor activity, characterized in that they contain, as active ingredient, at least one compound having an energy-rich bond chosen from a phosphoric amide bond or an enol phosphate bond, in combination with a pharmaceutical excipient acceptable.   6 Pharmaceutical compositions according to claim 4 or 5 characterized in that they contain, as active ingredient, a compound chosen from   creatine phosphate and phospho-enol-pyruvic acid.   7 A method of preparing a pharmaceutical composition, in particular with anti-tumor activity, characterized in that it consists in incorporating a pharmaceutically effective amount of at least one compound having an energy-rich bond chosen from a phosphoric amide bond or a phosphate bond   enol, in a pharmaceutically acceptable excipient.   8 Method according to claim 7, characterized in that the composition   pharmaceutical is formulated as an injectable preparation dosed from 0 1 to 50 g.   9 Use of at least one compound having an energy-rich bond chosen from a phosphoric amide bond or an enol phosphate bond   for the preparation of a medicament for the treatment of tumors.